Circulating Exosomal miR-1290 for Diagnosis of Epithelial Ovarian Cancer.

The aim of the study was to develop a new diagnostic biomarker for identifying serum exosomal miRNAs specific to epithelial ovarian cancer (EOC) and to find out target gene of the miRNA for exploring the molecular mechanisms in EOC. A total of 84 cases of ovarian masses and sera were enrolled, comprising EOC (n = 71), benign ovarian neoplasms (n = 13). We detected expression of candidate miRNAs in the serum and tissue of both benign ovarian neoplasm group and EOC group using real-time polymerase chain reaction. Immunohistochemistry were constructed using formalin fixed paraffin embedded (FFPE) tissue to detect expression level of suppressor of cytokine signaling 4 (SOCS4). In the EOC group, miRNA-1290 was significantly overexpressed in serum exosomes and tissues as compared to benign ovarian neoplasm group (fold change ≥ 2, p < 0.05). We observed area under the receiver operating characteristic curve (AUC) for miR-1290, using a cut-off of 0.73, the exosomal miR-1290 from serum had AUC, sensitivity, and specificity values of 0.794, 69.2 and 87.3, respectively. In immunohistochemical study, expression of SOCS4 in EOC was lower than that in benign ovarian neoplasm. Serum exosomal miR-1290 could be considered as a biomarker for differential diagnosis of EOC from benign ovarian neoplasm and SOCS4 might be potential target gene of miR-1290 in EOC.

Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance.

The role of epithelial-to-mesenchymal transition (EMT) in metastasis is a longstanding source of debate, largely owing to an inability to monitor transient and reversible EMT phenotypes in vivo. Here we establish an EMT lineage-tracing system to monitor this process in mice, using a mesenchymal-specific Cre-mediated fluorescent marker switch system in spontaneous breast-to-lung metastasis models. We show that within a predominantly epithelial primary tumour, a small proportion of tumour cells undergo EMT. Notably, lung metastases mainly consist of non-EMT tumour cells that maintain their epithelial phenotype. Inhibiting EMT by overexpressing the microRNA miR-200 does not affect lung metastasis development. However, EMT cells significantly contribute to recurrent lung metastasis formation after chemotherapy. These cells survived cyclophosphamide treatment owing to reduced proliferation, apoptotic tolerance and increased expression of chemoresistance-related genes. Overexpression of miR-200 abrogated this resistance. This study suggests the potential of an EMT-targeting strategy, in conjunction with conventional chemotherapies, for breast cancer treatment.

Heparin-Mimicking Polymer-Based In Vitro Platform Recapitulates In Vivo Muscle Atrophy Phenotypes.

The cell-cell/cell-matrix interactions between myoblasts and their extracellular microenvironment have been shown to play a crucial role in the regulation of in vitro myogenic differentiation and in vivo skeletal muscle regeneration. In this study, by harnessing the heparin-mimicking polymer, poly(sodium-4-styrenesulfonate) (PSS), which has a negatively charged surface, we engineered an in vitro cell culture platform for the purpose of recapitulating in vivo muscle atrophy-like phenotypes. Our initial findings showed that heparin-mimicking moieties inhibited the fusion of mononucleated myoblasts into multinucleated myotubes, as indicated by the decreased gene and protein expression levels of myogenic factors, myotube fusion-related markers, and focal adhesion kinase (FAK). We further elucidated the underlying molecular mechanism via transcriptome analyses, observing that the insulin/PI3K/mTOR and Wnt signaling pathways were significantly downregulated by heparin-mimicking moieties through the inhibition of FAK/Cav3. Taken together, the easy-to-adapt heparin-mimicking polymer-based in vitro cell culture platform could be an attractive platform for potential applications in drug screening, providing clear readouts of changes in insulin/PI3K/mTOR and Wnt signaling pathways.

Downregulation of glypican-4 facilitates breast cancer progression by inducing cell migration and proliferation.

Glypican-4 (GPC-4) is a heparan sulphate glycoprotein, associated with cell membrane via a Glycosyl phosphatidyl (GPI)-anchor. It is involved in cell migration, cell growth, differentiation and morphogenesis as well as chemoresistance and cancer stem cell maintenance in pancreatic cancer. However, its role in breast cancer remains unclear. To elucidate the role of GPC-4 in breast cancer, we analyzed GPC-4 expression in breast cancer patients and breast cancer cell lines. Our results demonstrated that GPC-4 expression was downregulated in metastatic tumors as compared to non-metastatic tumors. Further, GPC4's downregulation was confirmed in breast cancer metastatic cells (MDA-MBA-231 and MDA-MB-LM2) compared to non-metastatic cells (T47-D and MCF-7) with quantitative PCR and western blot. Knock-down of GPC-4 in non-metastatic cells significantly increased cell-migration and invasion. Similarly, over-expressing GPC-4 in metastatic cells decreased cell-migration/invasion and cell proliferation. Additionally, GPC-4 overexpression decreased in-vivo tumorigenicity in nude mice. Therefore, this research for the first time, has established the role of glypican-4 as a tumor-suppressor in breast cancer by decreasing migration and proliferation, revealing it as a possible therapy for breast cancer.

The circulating exosomal microRNAs related to albuminuria in patients with diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN) is associated with high risk of cardiovascular disease and mortality. Exosomal microRNAs (miRNAs) regulate gene expression in a variety of tissues and play important roles in the pathology of various diseases. We hypothesized that the exosomal miRNA profile would differ between DN patients and patients without nephropathy. METHODS: We prospectively enrolled 74 participants, including healthy volunteers (HVs), diabetic patients without nephropathy, and those with DN. The serum exosomal miRNA profiles of participants were examined using RNA sequencing. RESULTS: The expression levels of 107 miRNAs differed between HVs and patients without DN, whereas the expression levels of 95 miRNAs differed between HVs and patients with DN. Among these miRNAs, we found 7 miRNAs (miR-1246, miR-642a-3p, let-7c-5p, miR-1255b-5p, let-7i-3p, miR-5010-5p, miR-150-3p) that were uniquely up-regulated in DN patients compared to HVs, and miR-4449 that was highly expressed in DN patients compared to patients without DN. A pathway analysis revealed that these eight miRNAs are likely involved in MAPK signaling, integrin function in angiogenesis, and regulation of the AP-1 transcription factor. Moreover, they were all significantly correlated with the degree of albuminuria. CONCLUSIONS: Patients with DN have a different serum exosomal miRNA profile compared to HVs. These miRNAs may be promising candidates for the diagnosis and treatment of DN and cardiovascular disease.

mirRICH, a simple method to enrich the small RNA fraction from over-dried RNA pellets.

Techniques to isolate the small RNA fraction (<200nt) by column-based methods are commercially available. However, their use is limited because of the relatively high cost. We found that large RNA molecules, including mRNAs and rRNAs, are aggregated together in the presence of salts when RNA pellets are over-dried. Moreover, once RNA pellets are over-dried, large RNA molecules are barely soluble again during the elution process, whereas small RNA molecules (<100nt) can be eluted. We therefore modified the acid guanidinium thiocyanate-phenol-chloroform (AGPC)-based RNA extraction protocol by skipping the 70% ethanol washing step and over-drying the RNA pellet for 1 hour at room temperature. We named this novel small RNA isolation method "mirRICH." The quality of the small RNA sequences was validated by electrophoresis, next-generation sequencing, and quantitative PCR, and the findings support that our newly developed column-free method can successfully and efficiently isolate small RNAs from over-dried RNA pellets.

Carbon monoxide regulates glycolysis-dependent NLRP3 inflammasome activation in macrophages.

Low dose of carbon monoxide (CO) has anti-inflammatory role through various signaling pathways. Cellular metabolism has been implicated in the activation of inflammation in immune cells. However, the mechanisms by which CO-dependent metabolic regulation affect the immune response remain unclear. Here we show that CO-dependent metabolic pathway regulates the activation of the nucleotide-binding domain, leucine-rich-repeat-containing receptor (NLR), pyrin-domain-containing 3 (NLRP3) inflammasome. CO-releasing molecule-3 (CORM-3) resulted in reduced glycolysis-dependent NLRP3 inflammasome activation in macrophages. The reduced mTORC1 activation by CORM-3 resulted in less glycolysis during NLRP3 inflammasome activation. CORM-3 suppressed caspase-1 activation and the secretion of interleukin (IL)-1ß and IL-18 in macrophages in response to lipopolysaccharide (LPS) and ATP. Moreover, CORM-3 inhibits the oligomerization of the adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), which is required for NLRP3-dependent caspase-1 activation. Furthermore, CORM-3-treated mice showed substantial reduction in IL-1ß production by hyperglycemia in a mouse model of streptozotocin (STZ)-induced diabetes. Our results suggest that CO regulates glycolysis-dependent NLRP3 inflammasome activation and may provide a therapeutic approach for inflammation in metabolic diseases.

Effective experimental validation of miRNA targets using an improved linker reporter assay.

miRNAs are small, non-coding RNAs that play critical roles in various cellular processes. Although there are several algorithms that can predict the potential candidate genes that are regulated by a miRNA, these algorithms require further experimental validation in order to demonstrate genuine targets of miRNAs. Moreover, most algorithms predict hundreds to thousands of putative target genes for each miRNA, and it is difficult to validate all candidates using the whole 3'-untranslated region (UTR) reporter assay. We report a fast, simple and efficient experimental approach to screening miRNA candidate targets using a 3'-UTR linker assay. Critically, the linker has only a short miRNA regulatory element sequence of approximately 22 base pairs in length and can provide a benefit for screening strong miRNA candidates for further validation using the whole 3'-UTR sequence. Our technique will provide a simplified platform for the high-throughput screening of miRNA target gene validation.

Kidney Cancer and Potential Use of Urinary Extracellular Vesicles.

Kidney cancer is the 14th most common cancer globally. The 5-year relative survival rate of kidney cancer at a localized stage is 92.9% and it declines to 17.4% in metastatic stage. Currently, the most accurate method of its diagnosis is tissue biopsy. However, the invasive and costly nature of biopsies makes it undesirable in many patients. Therefore, novel biomarkers for diagnosis and prognosis should be explored. Urinary extracellular vesicles (uEVs) are small vesicles (50-200 nm) in urine carrying nucleic acids, proteins and lipids as their cargos. These uEVs' cargos can provide non-invasive alternative to monitor kidney health. In this review, we have summarized recent studies investigating potential use of uEVs' cargos as biomarkers in kidney cancer for diagnosis, prognosis and therapeutic intervention.

Characterization of exosomal microRNAs in preterm infants fed with breast milk and infant formula.

Breastfeeding not only reduces infection-related morbidity, but also increases growth of preterm infants. Advantages of breast milk (BM) for preterm infants are significant. They continue to be studied. However, because not all preterm infants can receive breastfeeding, bovine-based infant formula (IF) is used as an alternative, which may increase the risk of several preterm complications. Exosomes isolated from biofluids are emerging as biomarkers in research of various diseases. Here, we characterized miRNA contents of exosomes in urine and serum samples of preterm infants who were BM and IF fed and performed transcriptomic analysis of small RNA libraries. We identified significantly up-regulated 6 miRNAs and 10 miRNAs, respectively. Gene Ontology (GO) analysis revealed that target genes of these miRNAs might participate in neuronal development, immunity modulation, detoxification of reactive oxygen species, and transmembrane exchange. Our data suggest that exosome-based systemic screening for preterm infants with breastfeeding might be a screening tool for identifying target molecules involved in therapy for preterm infants in neonatal intensive care unit (NICU) and for future application as nutraceutical formulations or pharmaceuticals.

Apolipoprotein L1 is a tumor suppressor in clear cell renal cell carcinoma metastasis.

The 5-year survival rate of kidney cancer drops dramatically from 93% to 15% when it is metastatic. Metastasis constitutes for 30% of kidney cancer cases, in which clear cell renal cell carcinoma (ccRCC) is the most prominent subtype. By sequencing mRNA of ccRCC patient samples, we found that apolipoprotein L1 (APOL1) was highly expressed in tumors compared to their adjacent normal tissues. This gene has been previously identified in a large body of kidney disease research and was reported as a potential prognosis marker in many types of cancers. However, the molecular function of APOL1 in ccRCC, especially in metastasis, remained unknown. In this study, we modulated the expression of APOL1 in various renal cancer cell lines and analyzed their proliferative, migratory, and invasive properties. Strikingly, APOL1 overexpression suppressed ccRCC metastasis both in vitro and in vivo. We then explored the mechanism by which APOL1 alleviated ccRCC malignant progression by investigating its downstream pathways. APOL1 overexpression diminished the activity of focal adhesive molecules, Akt signaling pathways, and EMT processes. Furthermore, in the upstream, we discovered that miR-30a-3p could inhibit APOL1 expression. In conclusion, our study revealed that APOL1 play a role as a tumor suppressor in ccRCC and inhibit metastasis, which may provide novel potential therapeutic approaches for ccRCC patients.

Febrile Seizure Causes Deficit in Social Novelty, Gliosis, and Proinflammatory Cytokine Response in the Hippocampal CA2 Region in Rats.

Febrile seizure (FS), which occurs as a response to fever, is the most common seizure that occurs in infants and young children. FS is usually accompanied by diverse neuropsychiatric symptoms, including impaired social behaviors; however, research on neuropsychiatric disorders and hippocampal inflammatory changes following febrile seizure occurrences is very limited. Here, we provide evidence linking FS occurrence with ASD pathogenesis in rats. We developed an FS juvenile rats model and found ASD-like abnormal behaviors including deficits in social novelty, repetitive behaviors, and hyperlocomotion. In addition, FS model juvenile rats showed enhanced levels of gliosis and inflammation in the hippocampal CA2 region and cerebellum. Furthermore, abnormal levels of social and repetitive behaviors persisted in adults FS model rats. These findings suggest that the inflammatory response triggered by febrile seizures in young children could potentially serve as a mediator of social cognitive impairments.

Exosomal miRNA Profile in Small-for-Gestational-Age Children: A Potential Biomarker for Catch-Up Growth.

Objective: The mechanism underlying postnatal growth failure and catch-up growth in small-for-gestational-age (SGA) children is poorly understood. This study investigated the exosomal miRNA signature associated with catch-up growth in SGA children. Methods: In total, 16 SGA and 10 appropriate-for-gestational-age (AGA) children were included. Serum exosomal miRNA was analyzed using next-generation sequencing (NGS). Exosomal miRNA was profiled for five SGA children with catch-up growth (SGA-CU), six SGA children without CU growth (SGA-nCU), and five AGA children. Results: Exosomal miRNA profiles were clustered into three clear groups. The exosomal miRNA expression profiles of the SGA-nCU group differed from those of the SGA-CU and AGA groups. In all, 22 miRNAs were differentially expressed between SGA-nCU and AGA, 19 between SGA-nCU and SGA-CU, and only 6 between SGA-CU and AGA. In both SGA-nCU and SGA-CU, miR-874-3p was upregulated and miR-6126 was downregulated. Therefore, these two miRNAs could serve as biomarkers for SGA. Compared with SGA-CU and AGA, miR-30c-5p, miR-363-3p, miR-29a-3p, and miR-29c-3p were upregulated in SGA-nCU, while miR-629-5p and miR-23a-5p were downregulated. These six miRNAs could be associated with growth failure in SGA-nCU children. Conclusions: SGA children without CU have a distinct exosomal miRNA expression profile compared with AGA and SGA children with CU. Exosomal miRNAs could serve as novel biomarkers for CU.

The diagnostic value of circulating tumor DNA in hepatitis B virus induced hepatocellular carcinoma: a systematic review and meta-analysis.

Background/Aim: New biomarkers are urgently needed to aid in the diagnosis of early stage hepatocellular carcinoma (HCC). We performed a meta-analysis on the diagnostic utility of circulating tumor DNA (ctDNA) levels in patients with hepatitis B virus-induced HCC. Methods: We retrieved relevant articles from PubMed, Embase, and the Cochrane Library up to February 8, 2022. Two subgroups were defined; one subset of studies analyzed the ctDNA methylation status, and the other subset combined tumor markers and ctDNA assays. Pooled sensitivity (SEN), specificity (SPE), positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and area under the summary receiver operating characteristic curve (AUC) were analyzed. Results: Nine articles including 2,161 participants were included. The overall SEN and SPE were 0.705 (95% confidence interval [CI], 0.629-0.771) and 0.833 (95% CI, 0.769-0.882), respectively. The DOR, PLR, and NLR were 11.759 (95% CI, 7.982-17.322), 4.285 (95% CI, 3.098-5.925), and 0.336 (0.301-0.366), respectively. The ctDNA assay subset exhibited an AUC of 0.835. The AUC of the combined tumor marker and ctDNA assay was 0.848, with an SEN of 0.761 (95% CI, 0.659-0.839) and an SPE of 0.828 (95% CI, 0.692-0.911). Conclusions: Circulating tumor DNA has promising diagnostic potential for HCC. It can serve as an auxiliary tool for HCC screening and detection, especially when combined with tumor markers.

Gardnerella vaginalis in Recurrent Urinary Tract Infection Is Associated with Dysbiosis of the Bladder Microbiome.

Recent studies on the urine microbiome have highlighted the importance of the gut-vagina-bladder axis in recurrent urinary tract infection (rUTI). In particular, the role of Gardnerella as a covert pathogen that activates E. coli in animal experiments has been reported. Herein, we conducted a human bladder microbiome study to investigate the effect of Gardnerella on rUTI. Urine 16S ribosomal RNA gene sequencing via transurethral catheterization was conducted in the normal control group (NC) (n = 18) and rUTI group (n = 78). The positive detection rate of Gardnerella species did not differ between the NC and rUTI groups (22.2% vs. 18.0%, p = 0.677). In addition, the Gardnerella-positive NC and Gardnerella-positive rUTI groups showed similar levels of microbiome diversity. The Gardnerella-positive group was categorized into three subgroups: the Escherichia-dominant group, Gardnerella-dominant group, and Lactobacillus-dominant group. All of the Escherichia-dominant groups were associated with rUTI. The Gardnerella-dominant or Lactobacillus-dominant groups expressed rUTI with symptoms when risk factors such as the degree of Gardnerella proliferation or causative agents of bacterial vaginosis were present. The presence of Gardnerella in the urine is considered to be related to rUTI depending on other risk factors. New guideline recommendations regarding antibiotic selection based on a novel method to detect the cause of rUTI may be required to reduce antibiotic resistance.

Clinical impact of serum exosomal microRNA in liver fibrosis.

BACKGROUND/AIM: We investigated alterations in the expression of serum exosomal miRNAs with the progression of liver fibrosis and evaluated their clinical applicability as biomarkers. METHODS: This study prospectively enrolled 71 patients who underwent liver biopsy at an academic hospital in Korea. Exosomes were extracted from serum samples, followed by next-generation sequencing (NGS) of miRNAs and targeted real-time quantitative polymerase chain reaction. A model was derived to discriminate advanced fibrosis based on miRNA levels and the performance of this model was evaluated. Validation of the effect of miRNA on liver fibrosis in vitro was followed. RESULTS: NGS data revealed that exosomal miR-660-5p, miR-125a-5p, and miR-122 expression were changed significantly with the progression of liver fibrosis, of which miR-122 exhibited high read counts enough to be used as a biomarker. The level of exosomal miR-122 decreased as the pathologic fibrosis grade progressed and patients with biopsy-proven advanced fibrosis had significantly lower levels of exosomal miR-122 (P < 0.001) than those without advanced fibrosis. Exosomal miR-122 exhibited a fair performance in discriminating advanced fibrosis especially in combination with fibrosis-4 score and transient elastography. In a subgroup of patients with a non-viral etiology of liver disease, the performance of exosomal miR-122 as a biomarker was greatly improved. Inhibition of miR-122 expression increased the proliferation of the human hepatic stellate cell line, LX-2, and upregulated the expression of various fibrosis related proteins. CONCLUSION: Exosomal miR-122 may serve as a useful non-invasive biomarker for liver fibrosis, especially in patients with non-viral etiologies of chronic liver disease.

The complete mitogenome of the Chionoecetes opilio (Crustacea: Decapoda: Oregoniidae) and its unique characteristics.

The complete mitochondrial genome of Chionoecetes opilio is a 16,067 bp long, circular molecule which contains 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), and 2 ribosomal RNA genes (rRNAs). Its gene contents and organization are generally similar to other majoid mitogenomes. However, the mitogenome shows unique characteristics; long terminal amino acids, loss or addition of 3 PCGs, a 1216 bp long putative D-loop region, and peculiar secondary structures of 5 tRNAs. The concatenated amino acid sequences of 13 PCGs were used to analyze the phylogenetic tree, which well supported the monophyly of brachyuran clades of Majoidea, Heterotremata, Thoracotremata, and Eubrachyura.

Therapeutic miRNA-Enriched Extracellular Vesicles: Current Approaches and Future Prospects.

Extracellular vesicles (EVs) are 50-300 nm vesicles secreted by eukaryotic cells. They can carry cargo (including miRNA) from the donor cell to the recipient cell. miRNAs in EVs can change the translational profile of the recipient cell and modulate cellular morphology. This endogenous mechanism has attracted the attention of the drug-delivery community in the last few years. EVs can be enriched with exogenous therapeutic miRNAs and used for treatment of diseases by targeting pathological recipient cells. However, there are some obstacles that need to be addressed before introducing therapeutic miRNA-enriched EVs in clinics. Here, we focused on the progress in the field of therapeutic miRNA enriched EVs, highlighted important areas where research is needed, and discussed the potential to use them as therapeutic miRNA carriers in the future.

Whole leukemia cell vaccines: Past progress and future directions.

It has long been recognized that allogeneic hematopoietic stem cell transplantation can reduce the risk of leukemia relapse by inducing the graft-versus-leukemia effect. However, allogeneic stem cell transplantation is also known to be able to cause graft-versus-host disease, which can cause considerable morbidity and even mortality in patients receiving allogeneic hematopoietic stem cell transplantation. Therefore, to elicit leukemia-specific immune responses without alloimmune reaction, the possibilities of active immunotherapy methods such as leukemia vaccines have been studied for decades. Among various types of leukemia vaccines, whole leukemia cell vaccines are known to be able to induce immune responses against multiple unknown antigens without the need for adoptive transfer of dendritic cells. In this review, we will discuss the past progress of whole leukemia cell vaccines, with a focus on strategies to enhance their immunogenicity. We will also present the future directions of whole leukemia cell vaccines along with addressing newly emerging concepts, such as immunogenic cell death and necroptosis. We will not discuss in detail other factors that can reduce the therapeutic efficacy of whole leukemia cell vaccines such as various immunosuppressive mechanisms of leukemia.

Exosomes in Food: Health Benefits and Clinical Relevance in Diseases.

Exosomes are membrane-bound organelles generally secreted by eukaryotic cells that contain mRNAs, microRNAs, and/or proteins. However, recent studies have reported the isolation of these particles from foods such as lemon, ginger, and milk. Owing to their absorption by intestinal cells and further travel via the bloodstream, exosomes can reach distant organs and affect overall health in both infants and adults. The potential role of food-derived exosomes (FDEs) in alleviating diseases, as well as in modulating the gut microbiota has been shown, but the underlying mechanism is still unknown. Moreover, exosomes may provide biocompatible vehicles for the delivery of anti-cancer drugs, such as doxorubicin. Thus, exosomes may allow medical nutritionists and clinicians to develop safe and targeted therapies for the treatment of various pathologies. The present review introduces FDEs and their contents, highlights their role in disease and infant/adult health, and explores their potential use as therapeutic agents.